This project is designed to define the morphologic, molecular, and metabolic characteristics of breast ducts and ductal epithelial cells at normal risk and at high risk for breast cancer among Caucasian, Hispanic and African American women. This information is needed to define the early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, to identify new targets and to facilitate selection and monitoring of women for breast cancer prevention, and to define the molecular basis for disparities in the development and presentation of breast cancer. This project includes the following clinical and laboratory studies: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). Protocol 02-C-0077 characterizes by ductal lavage and ductal endoscopy the breast ducts and ductal epithelium of Caucasian, African American, and Hispanic women at normal risk and at high risk for breast cancer. One hundred forty six have been studied, 69 high risk subjects and 77 subjects at normal risk. A significantly improved method of ductal epithelial cell sampling has been developed under this study which provides multiple samples of pure (90%) ductal epithelial cells with high cellularity. These important findings were recently published (Danforth, DN et al, Breast Cancer: Basic and Clinical Research, 9:31-40, 2015). Extraction of a single intact ductal lavage sample (fluid and epithelial cells) or the separate frozen cellular component provided DNA and RNA suitable for multiple downstream molecular studies including whole genome DNA amplification, arrayCGH analysis, microarray gene expression profiling, RT-PCR of miRNA species, and qPCR of the telomerase gene. This method significantly expands our ability to define the molecular characteristics according to breast cancer risk of breast ductal epithelium in women at different risks for breast cancer, and also introduces a much needed method for collecting ductal epithelial cells from women at normal risk for breast cancer, a critical control group for defining the multiple molecular characteristics of women at increased risk for breast cancer. It has been shown that 50% - 70% of women in the U.S. who develop breast cancer have no identifiable risk factors, and thus our findings should also permit us to further characterize genomic changes and identify at-risk women in this group, a major population of women in the U.S. To compliment and guide the molecular studies in this protocol, a comprehensive literature review was conducted to identify and define genomic changes in normal breast tissue at normal risk and at high risk for breast cancer (Danforth, DN. Breast Cancer: Basic and Clinical Research 10:109, 2016). This indicated that normal risk breast tissues (such as reduction mammoplasty) contain evidence of early breast carcinogenesis including loss of heterozygosity, DNA methylation of tumor suppressor and other genes, and telomere shortening. In normal tissues at high risk for breast cancer (such as normal breast tissue adjacent to breast cancer or the contralateral breast), these changes persist and are increased and accompanied by aneuploidy, increased genomic instability, a wide range of gene expression differences, development of large cancerized fields, and increased proliferation. A model for the carcinogenic pathway in normal risk and in high risk normal breast tissue is proposed in this publication. The studies conducted under our protocol have also provided important clarification of the clinical and prognostic role of cytologic atypia in women at risk for breast cancer. Cytologic studies of ductal cells under protocol 02-C-0077 has revealed the presence of atypical epithelial cells in 26.5% of normal risk and 22.9 % of high risk subjects. These subjects were further evaluated by ductal endoscopy and breast MRI, which did not indicate an associated atypical hyperplasia lesion in any subject. Gene expression profiling studies did not demonstrate any gene expression differences in subjects with atypia vs. those without atypia. On follow-up, 2 high risk subjects and no normal risk subjects developed invasive breast cancer. Together these findings suggest epithelial atypia identified by ductal lavage may be of prognostic significance in selected women at high risk, but not in normal risk subjects. To complement these studies a review of the molecular profile of atypical ductal hyperplasia of the breast was recently published (Danforth, DN. Breast Cancer Res Treat, 167:9 2018). The gene expression studies also demonstrated the presence of intraepithelial lymphocytes in the ductal lavage cellular samples, with gene expression of CD3, CD4, CD8, B2M, Lck and GZM genes. Because intraepithelial lymphocytes are located primarily in the lobules in the proximal aspect of the duct, these findings indicate that the entire ductal contents are collected with the lavage sample, allowing for a comprehensive analysis of the ductal fluid, lymphocyte and epithelial cellular content. Studies are in progress to define the qualitative and quantitative characteristics of the lymphocyte population in the breast ducts of the normal risk and high risk populations. We are also developing in this project a potentially very valuable resource, the development of normal breast epithelial cell lines from the ductal lavage samples from women at different risks for breast cancer. We have demonstrated that human mammary epithelial cells may be cultured from a single ductal lavage sample. Demographic data including detailed risk assessment information is available for each subject, allowing for the development of a panel of cell lines according to different risks for breast cancer. This will provide abundant material for molecular profiling of risk characteristics, facilitate analysis of metabolic properties including response to exogenous mitogens and inhibitory substances, promote identification of alterations in signaling pathways according to risk, and allow development of an in vitro model useful for evaluation of chemoprevention drugs. Lastly, to compliment our studies of the basis of racial disparities, a comprehensive review of the literature has recently been published proposing for the first time a model describing the relationship of biological and nonbiological factors to the initiation and development of the major disparities in breast cancer between African American and Caucasian women (Danforth, DN Breast Cancer Research, 15:208-220, 2013). This model identified multiple molecular differences in breast cancer between African American and Caucasian women, and these differences are the major drivers of the disparities in age of onset, more advanced stage, more aggressive histology, and worse survival in African American vs. Caucasian women. Multiple socioeconomic, reproductive and health care factors influence the outcome of the disparities through their influence on the breast cancer molecular characteristics. The ductal sampling method which we have developed is applicable to all ethnic groups and both normal risk and high risk women, and thus represent an excellent means to further define the molecular differences in breast tissue associated with the disparities among ethnic groups at risk for breast cancer.